CN102283910A - Chinese medicinal composition with anti-depression effect and preparation and preparation method thereof - Google Patents

Abstract

The invention discloses a Chinese medicinal composition with an anti-depression effect. The Chinese medicinal composition is prepared from the following extracts in part by weight: 1 to 10 parts of white paeony root extract and 1 to 10 parts of bupleurum extract. The invention also discloses a preparation from the Chinese medicinal composition and a preparation method for the Chinese medicinal composition. In the technical scheme, the Chinese medicinal composition is obvious in a curative effect, high in purity and small in administration dose and has fewer side reactions; bupleurum total saponin and white paeony root total saponin are extracted by a modern pharmaceutic process, and depression relieving capsules prepared by compatibility in a certain ratio have definite medicinal effective parts and a definite curative effect; and the preparation method is simple, easy to operate, basically environment-friendly and suitable for industrial production, and greater social and economic benefit can be created.

Description

Traditional Chinese medicine composition with antidepressant effect, its preparation and preparation method

technical field

The invention belongs to the field of traditional Chinese medicine, and in particular relates to a composition of effective part of traditional Chinese medicine with antidepressant effect, a traditional Chinese medicine preparation containing the composition, and a preparation method of the composition.

Background technique

Depression is a group of mood disorders (Mood Disorders) or affective disorders (Affective Disorders) caused by various reasons with depression as the main symptom. It is a group of clinical symptoms or states centered on self-experience of depressed mood. , is an unpleasant mood experience. Depression is a common psychiatric disease that affects the body, emotions, and thinking. It seriously affects people's eating and sleeping patterns; it affects how individuals feel about themselves and how they think about problems. Symptoms are: loss of pleasure, decreased energy, psychomotor retardation or agitation, low self-esteem or guilt, decreased thinking ability, suicidal tendencies or self-injurious behavior, sleep disturbance, loss of appetite and libido and other symptoms.

According to the "2002 World Health Organization Report" issued by the World Health Organization, depression has ranked fourth among the top ten diseases in the world, and it is expected to jump to the second place by 2020, after myocardial infarction and before cancer. The figures of WHO show that there are 85 million depression patients in the world at present, and its incidence rate is expected to reach 10% of the total population by 2005. According to the latest data, the ranking of mental disorders in the total burden of disease has jumped to the first place, and the depression patients in Shanghai have accounted for more than 5% of the total population. Depression costs up to $150 billion a year in North America and the European Union. At the same time, the suicide rate of depressed patients is as high as 15%. However, due to lack of awareness, the vast majority of depression patients have not received correct diagnosis and treatment. Therefore, it is of great significance to enhance the understanding of depression, strengthen the research of antidepressants, and ensure the healthy development of society, and will produce greater economic and social benefits.

Treatment for depression can be divided into medication, psychotherapy, social therapy, and behavioral therapy. The high-incidence and difficult-to-cure depression is gradually building a considerable antidepressant market, which is one of the main reasons for the particularly active research and development of central nervous system drugs at home and abroad in recent years. According to statistics, antidepressant drugs accounted for more than 40% of the central nervous system drug market in the United States in 2000. The sales of the antidepressant drug market was 3 billion US dollars in 1995 and 6 billion US dollars in 1998, showing a sharp upward trend year by year. In 2000, among the top 10 therapeutic drugs in the world drug market sales, the annual sales of antidepressants reached 13.4 billion US dollars, accounting for 4.2% of the global sales share, ranking third; the sales growth rate was 18%, ranking first. number 5. In China, a survey on the use of antidepressants in eight major cities including Beijing, Shanghai, Guangzhou, Chongqing, Wuhan, Nanjing, Chengdu, and Hangzhou from 1997 to 2002 shows that the market for antidepressants in my country is growing rapidly. It can be predicted that the potential of the antidepressant drug market in the future will be quite large. Since 2001, the World Health Organization has taken attention to mental illness as one of its main tasks, and the Chinese health department has also listed depression as the focus of future prevention and treatment.

Clinically antidepressants are mostly treated with chemical drugs. Currently commonly used monoamine oxidase inhibitors (MAGI), such as phenelzine, phenprohydrazine and so on. Tricyclic antidepressants (TCAs), such as desipramine, amitriptyline, nortriptyline, etc. Heterocyclic antidepressants (HCA), such as maprotiline, mianserin, etc. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, paroxetine, sertraline, citalopram, etc. Serotonin and norepinephrine reuptake inhibitors (SNRI), venlafaxine, duloxetine, milnacipran, etc. Norepinephrine and specific serotonin reuptake inhibitors (NaSSAs), such as mirtazapine. In addition to the above categories, there are many drugs with certain antidepressant effects that are used clinically, such as drugs that adjust the conduction function of various central neurotransmitters, serotonin receptor blockers, and dopamine reuptake blockers. etc. At the same time, some antipsychotics also have antidepressant effects and can be used as adjuvant therapy.

Although the antidepressant prospect of Western medicine is good, the complexity of the pathogenesis and the singleness of the medication make the antidepressant treatment in an obvious asymmetrical situation. The existing problems of antidepressant drug treatment are: first, it cannot be effective for some refractory depression; Withdrawal syndrome is not conducive to long-term use; third, the onset is slow, and it takes about 2-3 weeks to produce results, which is not conducive to seizing the opportunity, controlling symptoms, and making patients with poor compliance lose confidence; fourth, depression The recurrence rate after cure is still high. Therefore, the research and development of antidepressants at home and abroad pay more and more attention to natural medicines, especially traditional Chinese medicines with a history of more than two thousand years.

At present, there are more ancient prescriptions Xiaoyao San, Sini San, Chaihu Guizhi Decoction, Chaihu Shugan San, Da Chaihu Decoction, Pinggan Kaiyu Decoction, Xuanyu Tongjing Decoction, etc., which have clear antidepressant effects. Dashen Decoction, Jieyu Hehuan Decoction, Chai Shao Liu Jun Zi Decoction, Chai Gui Wen Dan Ding Zhi Decoction, etc., all use Bupleurum and Bai Shao as the main ingredients in the prescription. According to statistics, among the 42 antidepressant prescriptions in "Treatise on Febrile Diseases", Bupleurum appears 23 times, and Peony appears 16 times. They are single herbs with the highest frequency of soothing the liver, relieving depression and promoting blood circulation. Traditional Chinese medicine believes that the common TCM syndrome types of depression are: liver depression and qi stagnation syndrome, liver depression and spleen deficiency syndrome, liver depression and phlegm obstruction syndrome, heart and spleen deficiency syndrome, and liver depression and blood stasis syndrome. Among them, the most common disease is liver-qi stagnation syndrome. It can be seen that choosing Bupleurum and Baishao as the basic prescription of antidepressant traditional Chinese medicine has clinical significance.

Contents of the invention

In order to overcome the defects of many adverse reactions, slow onset and high relapse rate of Western medicine for the treatment of depression, one of the purposes of the present invention is to provide a Chinese medicine effective part composition and Chinese medicine preparation with antidepressant effect, which has remarkable curative effect and high purity. , small dosage, less side effects.

The second object of the present invention is to provide a preparation method of the above-mentioned traditional Chinese medicine composition, which has stable process, feasible operation, high extraction rate, high purity, low environmental pollution, and is suitable for industrial production.

In order to achieve the above-mentioned purpose, the present invention has adopted following technical scheme:

The traditional Chinese medicine composition with antidepressant effect is prepared from the following extracts in parts by weight: 1-10 parts of Radix Paeoniae Alba extract and 1-10 parts of Bupleurum bupleuri extract.

Preferably, the Radix Paeoniae Alba extract is 9 parts, and the Radix Bupleurum extract is 1 part.

A traditional Chinese medicine preparation is made from the traditional Chinese medicine composition as described above, and its dosage form is any one of oral liquid, tablet, capsule, drop pill and granule, preferably capsule.

A traditional Chinese medicine preparation, which is prepared by adding equal weight of starch to the above traditional Chinese medicine composition, mixing evenly, and filling it in capsules. The content of paeoniflorin in the capsules must not be less than 7.50%, and the content of total saponins of paeony must not be less than 7.5%. less than 25.0%, the content of saikosaponin d shall not be less than 0.50%, and the content of total saikosaponins shall not be less than 2.50%.

A preparation method of a Chinese medicine composition, comprising the steps of:

1) Preparation of Radix Paeoniae Alba Extract: Take Radix Paeoniae Alba, add 8 times the amount of 70% ethanol respectively, extract 3 times, each time for 2 hours, recover ethanol from the obtained extract under reduced pressure, concentrate to 2.0g crude drug/ml, and take 1BV volume The concentrated solution is passed through the AB-8 macroporous adsorption resin at a flow rate of 1BV/hour, and after standing for 2 hours, it is washed with deionized water at a flow rate of 1BV/hour until the effluent is colorless, and then washed with 10BV 70% ethanol at 2BV/ Carry out elution at a speed of one hour, collect the eluate, recover ethanol under reduced pressure, concentrate to extract, vacuum dry at 60°C, take out, pulverize, and obtain Radix Paeoniae Alba extract;

2) Preparation of Bupleurum extract: take Bupleurum decoction pieces, add 10 times the amount of 80% ethanol containing 1.0% sodium carbonate respectively, heat and reflux extraction for 3 times, each time for 1.5 hours, and combine the extracts; the obtained extracts are recovered under reduced pressure Ethanol, concentrated to 0.2g crude drug/ml, measure 1BV extract, pass through AB-8 macroporous adsorption resin at a flow rate of 1BV/hour, let it stand for 2 hours, and elute with 10BV of deionized water at a flow rate of 1BV/hour , discard the eluent, then wash with 20% ethanol 10BV at a flow rate of 1BV/hour, discard the cleaning solution, and use 70% ethanol 10BV to elute with a speed of 2BV/hour, collect the eluent, and reclaim ethanol under reduced pressure, Concentrate to extract, vacuum dry at 60°C, take out, pulverize to obtain Bupleurum extract;

3) Preparation of composition: according to the weight ratio of Radix Paeoniae Alba extract:Bupleurum extract 9:1, the above two extracts were weighed and mixed uniformly to prepare the above-mentioned traditional Chinese medicine composition.

Among them, "70% ethanol" is an ethanol solution with a concentration of 70%, "80% ethanol" and "20% ethanol" are the same, "80% ethanol containing 1.0% sodium carbonate" is an ethanol solution with a concentration of 80% The solution contains 1.0% sodium carbonate by weight; "6 times the amount of 70% ethanol" is a 70% ethanol solution whose weight is 6 times the weight of the decoction pieces, and "8 times the amount and 10 times the amount" is the same.

The Radix Paeoniae Alba used in the present invention is the dry root of Paeonia lactiflora Pall., a plant of the Ranunculaceae family. It is good at nourishing yin and blood, and can soften the liver and calm the liver, relieve spasm and relieve pain, and clear away asthenic heat. Bupleurum is the dry root of Bupleurum scorzonerifolium Willd., light and clear, good at loosening and walking, pungent and good at doing, it is a good product for soothing the liver and regulating qi, opening depression and reducing fever. The softness can promote the liver qi without dredging too much, which consumes the body of the liver; the powder of Bupleurum from white peony root can nourish the liver blood without causing stagnation and stagnation of qi and yin, which hinder the function of the liver. merit. It can not only soothe the liver and relieve stagnation, so as to treat the insufficiency of the liver, but also soften the liver and replenish yin to nourish the liver and body. It is the best combination for both body and function.

The invention extracts total saponins of Bupleuri and total saponins of paeony through modern pharmaceutical technology, and makes Jieyu capsules according to a certain compatibility ratio, has clear medicinal effective parts and clear curative effects, simple preparation method, feasible operation, and less environmental pollution , suitable for industrial production, and can achieve better social and economic benefits.

Detailed ways

The following is the analysis and experiment of each factor in the effective part composition of traditional Chinese medicine for antidepressant effect and its preparation method:

1. Preparation of Radix Paeoniae Alba Extract:

1. Optimization of Extraction Process of Total Saponins of Paeoniae Alba

The active ingredients of Radix Paeoniae Alba are monoterpenoids such as paeoniflorin, paeoniflorin alba, oxidized paeoniflorin and paeoniflorin benzophthalein, referred to as total saponins of paeony. Factors affecting the extraction of total saponins of Paeoniae Alba include ethanol concentration, ethanol dosage (volume to weight ratio, that is, the ratio of ethanol volume to medicinal material weight), extraction time, and extraction times. This experiment adopts L ₁₈ (3 ⁷ ) orthogonal design and single factor Investigate the combined method, measure the content of total saponins of white paeony and paeoniflorin in the extract of Radix Paeoniae Alba, and take the weighted fraction of total saponins of paeony and paeoniflorin as the evaluation index, optimize the extraction process of total saponins of paeony, see table 1 to Table 5.

Table 1 Paeony saponin extraction experimental design table

Table 2 Experiment plan

According to the above experimental design, extract, filter, combine the filtrates, and dilute to a certain volume. The content of total saponins and paeoniflorin in the extract was determined, and the results are shown in Table 3.

Table 3 Contents of total saponins of paeony and paeoniflorin in each extract

The content of total saponins of paeony and paeoniflorin is used as the index to carry out weighted scoring, and the formula is as follows:

将分值进行正交分析见表4：

Orthogonal analysis of the scores is shown in Table 4:

Table 4 visual analysis table

Table 5 variance analysis table

The result shows that there is a significant difference in the number of extractions. From the visual analysis table, the ethanol concentration is 70%, and the consumption is 8 times the amount, and the extraction effect is the best for each 2 hours. Therefore, this condition is fixed. The comparison of 4 times shows that the effect of extraction 3 times is not much different from that of 4 times, so the number of times of extraction is determined to be 3 times.

Verification test: take 200g of Radix Paeoniae Alba decoction pieces, 3 parts in total, put them in a 10000ml round bottom flask, add 8 times the amount of 70% ethanol, extract 3 times for 3 parts, extract 4 times for 3 parts, 2 hours each time, filter, and combine the filtrates , the filtrate was settled to 6000ml, and the content of total saponins of paeony and paeoniflorin in the extract was measured. The results showed that the average content of paeoniflorin was 21.49mg/g, and the average content of total saponins of paeony was 42.90mg/g. Similar results to the best process (group 13).

Finally, the extraction process of total saponins of Paeoniae Alba was determined as follows: take decoction pieces of Paeoniae Alba, add 8 times the amount, 70% ethanol, extract 3 times, 2 hours each time.

2. Optimal purification process of total saponins of paeony

Investigation of Static Adsorption Capacity

Take by weighing 5.0g (dry weight) of large porous adsorption resin, resin types are respectively: DM-130, 860021, DM-2, DM-131, AB-8, D101, add 15ml Radix Paeoniae Alba extract (0.2g crude drug /ml), left for 24 hours. Suction filtration, filtrate constant volume to 25ml, measure the content of total saponins of paeony in the solution, the result shows, the static adsorption capacity of AB-8 macroporous adsorption resin is obviously better than other resins, so choose AB-8 macroporous adsorption resin.

Investigation of the maximum dynamic adsorption capacity

Take pretreated AB-8 macroporous adsorption resin 80g (wet weight), pack into a column (inner diameter 4cm, column height 26cm), get Radix Paeoniae Alba extract (2.0g crude drug/ml) 50ml, with 80ml/h (1BV ) at the speed of sample loading, and the effluent was collected. After standing for 2 hours, add deionized water to elute at a speed of 80ml/h, collect 100ml/part, and measure the content of total saponins of paeony in the effluent and eluent. Results The maximum dynamic adsorption capacity was equivalent to 68.27ml of sample solution, and 2ml of Radix Paeoniae Alba extract could be adsorbed per gram of dry resin (2.0g crude drug/ml).

Elution Preliminary Experiment

Use 200ml of 10%, 20%, 30%, 40%, 50%, and 70% ethanol to elute the above-mentioned AB-8 resin column of the Radix Paeoniae Alba extract at a speed of 80ml/h, and collect and elute with 50ml/part solution, and the content of total saponins of paeony in the eluate was determined.

The results showed that the 10%-70% ethanol eluent contained total saponins of paeony, which may be due to the poor adsorption capacity of AB-8 macroporous resin for total saponins of paeony. Basically the same, the reason may be that the total saponins of Paeoniae Alba have a wide range of polarity, and different concentrations of ethanol have a certain eluting ability. Considering that the extract of Paeoniae Alba is applied to the column, after removing impurities with water, directly use a certain concentration of ethanol Carry out elution, measure the content of total saponins of paeony in the eluent, if it reaches 60%, it shows that the method is feasible.

Get 80g (wet weight) of pretreated AB-8 macroporous adsorption resin, pack into a column (inner diameter 4cm, column height 20cm), get Radix Paeoniae Alba extract 80ml, load the sample at a speed of 80ml/h, collect the effluent, place After 2 hours, add deionized water for elution until the effluent is substantially colorless, then use 70% ethanol 200ml for elution at a speed of 80ml/h, collect the eluent at 50ml/part, measure the content of benzoic acid in the eluent, The results showed that the first eluate contained no total saponins of paeony, and the contents of total saponins of paeony in the remaining three eluates were all greater than 60%, so the method was feasible.

Inspection of loading speed

Get the pretreated AB-8 macroporous adsorption resin 80g (wet weight), totally three, get Radix Paeoniae Alba extract 100ml, load the sample with the speed of 80ml/h, 160ml/h, 240ml/h respectively, collect the effluent, After standing for 2 hours, add deionized water to elute with a speed of 80ml/h (1BV/h) until the color of the effluent does not change substantially, collect the eluent at 100ml/part, measure the content of benzoic acid in the effluent and the eluent . The results show that the adsorption capacity is the largest when the column loading speed is 80ml/h, so the column loading speed is determined to be 1BV/hour.

Investigation on the concentration of upper column solution

Take 100ml of Radix Paeoniae Alba extract (2.0g crude drug/ml), 4 parts in total, dilute 2, 5, 10, and 20 times respectively, load the sample at a speed of 80ml/hour, collect the effluent, leave it for 2 hours, add deionized Water 800ml is eluted with the speed of 80ml/h, and 100ml/ part is collected, and measures the content of benzoic acid in effluent and eluent. The results showed that the adsorption capacity of Radix Paeoniae Alba extracts with different concentrations was basically the same. In order to shorten the sample loading time, the concentration of the column solution was selected (2.0g crude drug/ml).

Investigation on the Concentration and Dosage of Eluent

Get pretreated AB-8 macroporous adsorption resin 80g (dry weight), three in total, take 80ml of Radix Paeoniae Alba extract respectively, load the sample at a speed of 80ml/hour, after standing for 2 hours, add deionized water at 80ml/hour The speed of elution of h until the color of the effluent is basically unchanged, and then eluted with 50%, 60%, and 70% ethanol at a speed of 80ml/h until the color of the effluent is basically unchanged, and the eluate is collected at 100ml/part, and measured The content of benzoic acid in the eluent. The results showed that 800ml of 70% ethanol, that is, 10BV, could completely elute the total saponins of paeony, and the elution rate was 90.40%, so it was determined to use 70% ethanol 10BV as the eluent.

The investigation of elution speed

Take 80ml of Radix Paeoniae Alba extract (2.0g crude drug/ml), a total of 3 parts, load the sample, and after standing for 2 hours, elute with deionized water until the color of the eluent does not change substantially, and use 70% ethanol 10BV to treat the three columns respectively. Carry out elution with the speed of 1BV/h, 2BV/h, 3BVh, 4BV/h, discard the first 100ml eluate liquid, all the other are collected with 200ml/ part, eluate, measure the content of benzoic acid in the eluate content. The results showed that the elution rates of 1BV/h and 2BV/h had little difference. Considering the higher efficiency of 2BV/h, the elution rate was determined to be 2BV/h.

Aspect-to-Height Ratio Inspection

Take three chromatographic columns, weigh 80g, 160g, 240g (wet weight) pretreated AB-8 macroporous adsorption resin respectively, pack them into columns, and take 80ml, 160ml, 240ml of Radix Paeoniae Alba extract (2.0g crude drug/ml) , load the sample at a speed of 1BV/h, after standing for 2 hours, elute with 10BV of water, 10BV of 20% ethanol, and then elute with 10BV of 70% ethanol, collect 70% ethanol eluate at 2BV/part, and measure the eluate content of benzoic acid. The results showed that the elution rates of the three diameter-to-height ratios were basically the same, indicating that the diameter-to-height ratio had little effect on the elution rate.

Verification experiment

Get the processed AB-8 macroporous adsorption resin, pack into columns (three, inner diameter 9.1cm, height 70cm, retention volume 1500ml), get 1500ml Radix Paeoniae Alba extract (2.0g crude drug/ml) loading sample with 1500ml/h respectively Load the sample at a high speed, after standing for 2 hours, elute with deionized water at a rate of 1500ml/hour, discard the eluent, elute with 15000ml of 70% ethanol at a rate of 3000ml/hour, collect the eluent, and recover under reduced pressure Ethanol, vacuum-dried, crushed to obtain 3 batches of total saponins of paeony, and the content was determined. The results showed that the yields of total saponins of paeony among the three batches were basically the same, the highest yield was 78.89%, and the contents of total saponins of paeony were also similar, so the repeatability of the experiment was good.

Table 6 verification experiment results

The final determination of the purification process of total saponins of Paeoniae Alba is as follows: take the pretreated AB-8 macroporous adsorption resin, pack it into a column, take 1BV Paeoniae Alba extract (2.0g crude drug/ml) and load it at a speed of 1BV/h, and place it for 2 Hours later, use deionized water to elute at a rate of 1BV/h until the eluate is colorless, discard the eluate, and use 70% ethanol 10BV to elute at a rate of 2BV/h, collect the eluate, and recover under reduced pressure Ethanol, concentrated to the extract, vacuum-dried at 60°C, taken out, crushed to obtain the Radix Paeoniae Alba extract.

2. Preparation of Bupleurum extract:

1. Optimization of extraction process of total saponins of Bupleurum chinensis

As a commonly used clinical medicine, Bupleurum bupleuri is mostly extracted from saikosaponins by decoction. Because the chemical structures of saikosaponins a and d are unstable, they are easily degraded by acid or heat to form secondary glycosides b1 with weaker activity. , b2, so with the prolongation of the extraction time, the amount of saikosaponin a, d will gradually decrease, affecting the quality and clinical efficacy of Bupleurum preparations. At present, there are many domestic literature reports on the extraction process of Bupleurum, but most of them use saikosaponin a alone as the index for investigation, and there are few literature reports on the simultaneous inspection of saikosaponin a, d and total saikosaponins. Therefore, the present invention adopts an orthogonal test to comprehensively investigate the contents of saikosaponins a, d and total saikosaponins in Bupleurum bupleuri, and screen the optimal process.

Investigation on Water Absorption and Alcohol Absorption

Take 50g of bupleurum decoction pieces, 2 parts in total, add 300ml of water to one part, and 300ml of 50% ethanol in the other part, place until it penetrates the heart, filter, and measure the water absorption rate and alcohol absorption rate. Results When extracting with water or 50% ethanol, 1.8L/Kg of water or 1.2L/Kg of 50% ethanol should be added respectively.

Investigation of extraction solvent

Take 50g of Bupleurum decoction pieces, a total of 2 parts, add 300ml of water to one part, add 300ml of 50% ethanol to one part, heat and reflux extraction 3 times, 1 hour each time, filter, combine the filtrates, dilute to 1000ml, measure the The content of total saponins of Bupleurum. The results showed that the extraction effect of 50% ethanol was better than that of water.

Examination of extraction methods

Take 50g of Bupleurum decoction pieces, 2 parts in total, soak them in 50% ethanol 300ml at 60°C, and extract them at 85°C for 3 times, each time for 1 hour, filter, combine the filtrates, and dilute to 1000ml, measure the Chaihu in the extract. Content of total saponins. The results showed that the heating and reflux extraction had the best extraction effect.

Influence of Adding Alkali on Extraction Effect

Referring to the relevant literature and the 2005 edition of the "Chinese Pharmacopoeia", it is found that a certain amount of alkali should be added during extraction, and the extraction with alkali and without alkali is compared.

Take 50g of Bupleurum decoction pieces, a total of 2 parts, one part without adding alkali, one part for extraction with 5% sodium carbonate, reflux extraction with 300ml of 50% ethanol for 3 times, each time for 1 hour, filter, combine the filtrates, and dilute to 1000ml. The content of total saponins in the extract was determined. The results showed that the content of total saponins increased after adding alkali, so a certain amount of alkali should be added during extraction.

Orthogonal Design to Investigate the Extraction Process of Bupleurum Total Saponins

Factors affecting the extraction of total saponins of Bupleurum include ethanol concentration, ethanol dosage, extraction time, extraction times and alkali concentration (dosage). This experiment adopts L ₁₈ (3 ⁷ ) Total saikosaponin was used as an index to investigate the influence of the above factors on the extraction of saikosaponin. See Tables 7-9 for the experimental design and results.

Table 7 Saikosaponin extraction experimental design table

Taking the content of total saikosaponins and saikosaponin d as indicators to carry out weighted scoring, the formula is as follows:

Table 8 visual analysis table

Table 9 variance analysis table

The results show that there is a significant difference between the concentration of ethanol and the number of times of extraction, so it is determined that the concentration of ethanol is 80%, and the number of times of extraction is 3 times. From the visual analysis table, it can be seen that there is little difference between the extraction time of 1.5 hours and 2 hours, so the extraction time is determined For 1.5 hours, with the increase of ethanol dosage, the contents of total saikosaponins and saikosaponin d increased, and the comparison should be made between 8 times and 10 times the amount of ethanol.

During the extraction process, it was found that the addition of 0.4% anhydrous sodium carbonate could not completely dissolve the 80% ethanol extraction, so the amount of alkali used in the 80% ethanol extraction should be investigated.

Investigation of Alkali Dosage

Weigh 50g of Bupleurum decoction pieces respectively, put them in a 1000ml round-bottomed flask, add 10 times the amount of 80% ethanol, and add a certain amount of anhydrous sodium carbonate (calculated by the volume of ethanol), and extract 3 times under reflux in a water bath at 80°C, each time 1.5 hour, filter, combine the filtrates, and extract the volume to 1500ml. The contents of total saikosaponins and saikosaponin d in the extract were determined. The results showed that the content of saikosaponin a and d reached the maximum when the alkali concentration was 1.0%, so the alkali concentration was determined to be 1.0%.

Investigation of ethanol consumption

Take 500g of Bupleurum decoction pieces, 2 parts in total, put them in a 10000ml round bottom flask, add 8 times the amount and 10 times the amount of 80% ethanol containing 1.0% anhydrous sodium carbonate respectively, heat and reflux for extraction 3 times, each time for 1.5 hours, Filtrate, combine the filtrates, set the volume of the filtrate to 15000ml, measure the content of total saikosaponins and saikosaponins a and d in the extract, and perform the experiment twice in parallel. The results showed that when the amount of ethanol was 10 times, the average content of saikosaponin a, d and total saikosaponin was higher than that of 8 times the amount of ethanol, so the amount of ethanol was determined to be 10 times.

Verification test

Take 500g of Bupleurum decoction pieces, 3 parts in total, put them in a 10L round bottom flask, add 10 times the amount of 80% ethanol and 1.0% sodium carbonate, heat and reflux for 3 times, each time for 1.5 hours, filter, and combine the filtrates, The filtrate was settled to 15000ml, and the content of total saikosaponins and saikosaponin a, d in the assay extract. Results The average content of saikosaponin a was 1.1221 mg/g, saikosaponin d was 4.1216 mg/g, and total saikosaponin was 50.64 mg/g. The result is close to that of the best process.

To sum up, the extraction process of total saponins of Bupleuri is determined as follows: take Bupleurum decoction pieces, add 10 times the amount of 80% ethanol containing 1.0% sodium carbonate, and extract 3 times, 1.5 hours each time.

2. Optimal purification process of total saponins of Bupleurum

The macroporous adsorption resin is used for purification. The factors affecting the purification effect of the macroporous adsorption resin include the type of macroporous adsorption resin, the concentration of the column solution, the column loading speed, the concentration and dosage of the eluent, and the elution speed.

Investigation of Static Adsorption Capacity

Take by weighing 5.0g (dry weight) of large porous adsorption resin, resin types are respectively: DM-130, 860021, DM-2, DM-131, AB-8, D101, add 15ml Bupleurum extract (0.8g crude drug /ml), left for 24 hours. Suction filtration, the filtrate was settled to 25ml, and the content of total saikosaponins in the solution was measured. The results show that the static adsorption capacity of AB-8 macroporous adsorption resin is obviously superior to other resins.

Investigation of the maximum dynamic adsorption capacity

Weigh 20g (dry weight) of the pretreated AB-8 resin macroporous adsorption resin, pack it into a column (inner diameter 4cm, column height 21cm), take 50ml of Bupleurum radix extract (0.8g crude drug/ml), add water to dilute to 200ml, Load the sample at a rate of 50ml/h (1BV/h), and collect the effluent. After standing for 2 hours, add deionized water to elute at a speed of 50ml/h, collect 100ml/part, and measure the content of total saponins in the effluent and eluent. The maximum dynamic adsorption capacity is equivalent to 177ml of sample solution, and each gram of dry resin can absorb 88.5ml of Bupleurum extract (0.8g crude drug/ml).

Elution Preliminary Experiment

The above resin column was eluted with 200ml of 10%, 20%, 30%, 40%, 50%, and 70% ethanol respectively at a speed of 50ml/h, and the eluate was collected at 50ml/part, and Bupleurum bupleuri in the eluate was measured. total saponin content. The results showed that the 10%-40% ethanol eluent did not contain Bupleurum saponins, and the 50% ethanol eluent contained Bupleurum saponins, but the content was less, indicating that 50% ethanol could effectively elute the total Bupleurum saponins. The ability is weak, and the eluent of 70% ethanol contains a large amount of total saikosides, indicating that 70% ethanol can be used as the eluent of total saikosaponins.

Investigation on the concentration of upper column solution

Take 50ml of Bupleurum extract (0.8g crude drug/ml), 4 parts in total, dilute 2, 4, 6, and 8 times respectively, load the sample at a speed of 50ml/hour (1BV/h), collect the effluent, and let it stand for 2 hours Afterwards, add deionized water 400ml to elute with the speed of 50ml/hour, 100ml/part is collected, measure the content of total saikosides in the effluent and the eluent. The results show that the adsorption capacity is the largest when the Bupleurum extract is diluted 4 times, that is, 0.2g crude drug/ml, and the concentration of the upper column solution is determined to be 0.2g crude drug/ml.

Investigation of the flow rate of upper column liquid

Take Bupleurum extract (0.2g crude drug/ml) 200ml, totally 4 parts, respectively with 50ml/h (1BV/h), 100ml/h (2BV/h), 150ml/h (3BV/h), 200ml/h (4BV/h) speed sample loading, collect the effluent, after standing for 2 hours, add deionized water 400ml to elute at a speed of 50ml/h, collect 100ml/part, measure the total saponins in the effluent and eluent content. The results show that when the flow rate of the upper column liquid is 50ml/h (1BV/h), the adsorption capacity is the largest, so the flow rate of the upper column liquid is determined to be 1BV/h.

Determination of concentration of impurity removal solution

It can be seen from the pre-elution experiment that water and 10%-40% ethanol can be used as impurity removal solutions. Take 150ml of Bupleurum extract (0.2g crude drug/ml) (80% of the maximum dynamic adsorption capacity) and put it on the sample. After standing for 2 hours, wash with water, 10% ethanol, 20% ethanol, 30% ethanol, and 40% ethanol respectively. Take off until the eluate is light yellow and transparent, basically unchanged, collect the eluate at 100ml/part, and measure the weight of the solids in the eluate. The result shows that after deionized water elutes 500ml (10BV), the color of the eluent does not change substantially, and the solids are less; 10% and 20% ethanol can remove certain impurities, while 30%, 40% ethanol removes impurities less. And the direct elution with 20% ethanol is investigated, the result shows that its solid matter is basically consistent with the total amount eluted with 10% and 20% ethanol respectively, so it is determined to use 20% ethanol for impurity removal, and 20% ethanol has been investigated. The consumption of ethanol, confirm consumption is 500ml (10BV).

Investigation on the Concentration and Dosage of Eluent

Take Bupleurum extract (0.2g crude drug/ml) 150ml, a total of 3 parts, load the sample, after standing for 2 hours, elute with 10BV of deionized water, 10BV of 20% ethanol, and wash with 60%, 70%, 80% ethanol respectively The three columns were eluted at a speed of 1BV/h until the eluate was substantially colorless, and the eluate was collected at 100ml/part (2BV/part), and the content of total saikosaponins in the eluate was measured. The result shows that 70% ethanol, 80% ethanol eluting 900ml (18BV) can wash out 90% of the total saponins of Bupleurum, and 1100ml (22BV) of 60% ethanol can only wash out the total saponins of 83.92%, so determine 70% ethanol was used as eluent. In 70% ethanol, the first 10BV can elute 83% of the total saikosaponins, so the amount of eluent is determined to be 10BV.

The investigation of elution speed

Take Bupleurum extract (0.2g crude drug/ml) 150ml, a total of 3 parts, load the sample, after standing for 2 hours, use deionized water 10BV, 20% ethanol 10BV to elute, respectively use 70% ethanol 10BV to three columns with 1BV The speed of /h, 2BV/h, 3BV/h, 4BV/h is eluted, and 100ml/ part (2BV/ part) collects eluate, measures the content of saikosaponin in eluate.

The results showed that the elution rate of 1BV/h was 86.96%, the elution rate of 2BV/h was 87.32%, the elution rate of 3BV/h was 79.81%, and the elution rate of 4BV/h The rate is 76.49%. The elution rate is the largest when the speed is 2BV/h, so the elution speed is determined to be 2BV/h.

Aspect-to-Height Ratio Inspection

Take three chromatography columns, weigh 20g, 40g, and 60g (dry weight) of pretreated AB-8 macroporous adsorption resin respectively, pack them into columns, and take 150ml, 300ml, and 450ml of Bupleurum extract (0.2g crude drug/ml) , loaded at a speed of 1BV/hour, after standing for 2 hours, eluted with 10BV of water and 10BV of 20% ethanol, and then eluted with 10BV of 70% ethanol, collected 70% ethanol eluate at 2BV/part, and measured the eluate The content of total saponins in Bupleurum. The results showed that the elution rates of the three diameter-to-height ratios were basically the same, indicating that the diameter-to-height ratio had little effect on the elution rate.

Verification experiment

Take the pretreated AB-8 macroporous adsorption resin, pack it into columns (three, inner diameter 9.1cm, height 70cm, retention volume 1500ml), take 10000ml Bupleurum extract (0.2g crude drug/ml) respectively, with the speed of 1500ml/h Load the sample, and after standing for 2 hours, use 15000ml of deionized water to elute at a speed of 1500ml/h, discard the eluent, then use 15000ml of 20% ethanol to elute at a speed of 1500ml/h, discard the eluent, and use 15000ml of 70% ethanol was eluted at a speed of 3000ml/h, the eluent was collected, the ethanol was recovered under reduced pressure, vacuum dried, pulverized to obtain 3 batches of total saponins of Bupleurum, and the content was determined. The results showed that the yields of the three batches of total saponins were basically the same, and the contents of the total saponins were also similar, and the repeatability of the experiment was good.

Table 10 verification experiment results

Finally, the purification process of Bupleurum saponins is determined as follows: take the pretreated AB-8 macroporous adsorption resin, pack it into a column, take 1BV of Bupleurum extract (0.2g crude drug/ml), and pass through AB-8 at a flow rate of 1BV/hour. Macroporous adsorption resin, after standing for 2 hours, eluted with 10BV of deionized water at a flow rate of 1BV/hour, discarded the eluent, and then washed with 10BV of 20% ethanol at a flow rate of 1BV/hour, discarded the cleaning solution, Elute with 70% ethanol 10BV at a rate of 2BV/hour, collect the eluate, recover ethanol under reduced pressure, concentrate to extract, vacuum-dry at 60°C, take out, and pulverize to obtain Bupleurum bupleuri extract.

Three, the preparation method of capsule:

According to the efficacy screening test, the total saponins of Paeoniae Alba-total saponins of Bupleurum (9:1) has the best drug effect, and the total saponins of Paeony and total saponins of Bupleurum are mixed uniformly according to the above ratio, and the bulk density and angle of repose are determined for it. Determination.

Bulk Density Determination

Take 10g of the above mixture, put it in a 50ml graduated cylinder, measure the volume, and calculate the bulk density. The results showed that the bulk density of this product was 0.8197g/ml.

Determination of angle of repose

The angle of repose of the mixed powder was measured, and the results are shown in Table 7.

Table 7 Total Saponin Mixture Angle of Repose Determination Result

The results show that the fluidity of the powder is better.

Determination of the amount of diluent

Each capsule of this product should contain 150mg of the mixture of total saponins of Bupleuri and total saponins of white paeony, and it is filled in No. 2 capsules. The volume of No. 2 capsule is 0.4ml, and the bulk density of starch is 0.6514g/ml, therefore, each capsule can also hold 150mg of starch. The total saponin mixture and starch were mixed uniformly (1:1), and the bulk density and angle of repose of the mixture were measured. The results show that the fluidity of this product is better, and starch can be used as a diluent of this product. The bulk density of this product is 0.7752g/ml, and each capsule is filled with 300mg. Choose No. 2 capsules for trial loading. As a result, it was determined that No. 2 capsules were used for subpackaging.

Critical Relative Humidity Determination

In order to prevent the powder from absorbing moisture, the critical relative humidity of the powder was measured to provide a reference for controlling the relative humidity in the packaging workshop.

Hygroscopic balance curve of Jieyu capsule contents

Determination method: Take 2 parts of the medicine powder, weigh them accurately, open the weighing bottle cap, put them into a glass desiccator with a humidity of 58%, place them in an incubator at 25°C, and store them at 1, 2, 3, 6, 9, 12, 24, 36, 48, and 72 hours Accurately weigh the weight of the weighing bottle and the powder, calculate the percentage of moisture absorption, and draw the moisture absorption balance curve. The results showed that after standing for 48 hours, the test product reached moisture absorption equilibrium.

Determination of critical relative humidity

Measuring method: Take 12 parts of the medicinal powder, divide them into 2 groups, 6 parts in each group, about 2g in each group, weigh them accurately, open the cap of the weighing bottle, put them into glass desiccators with different humidity respectively, and place them in a 25°C incubator Place it for 48 hours, weigh it accurately, and calculate the moisture absorption rate. The critical relative humidity curve is drawn, and the results show that the critical relative humidity of the medicinal powder is 63%, so when subpackaging, the relative humidity should be controlled below 60%.

Take three batches of total saponins of paeony, each 540g, three batches of total saponins of Bupleuri, each 60g, mix evenly, add 600g starch respectively, mix evenly, and fill in No. 2 capsules. The content of paeoniflorin in the capsule shall not be less than 7.50%, the content of total saponins of paeony shall not be less than 25.0%, the content of saikosaponin d shall not be less than 0.50%, and the content of total saikosaponins shall not be less than 2.50%.

Four, the composition formula ratio screening test of the present invention

1. Experimental materials

1.1 Drugs

The above-mentioned Bupleurum extract and the above-mentioned Radix Paeoniae Alba extract are suspended with 0.5% CMC-Na, homogenized by a high-speed homogenizer for 10 minutes, made into a suspension, and shaken before use;

Clomipramine hydrochloride tablets (Novartis Pharmaceuticals, batch number X0026, 25mg/tablet);

1.2 Experimental animals

ICR mice, clean grade. Half male and half female, provided by Zhejiang Experimental Animal Center, experimental animal qualification certificate number: Zheyi Dongzi No. 2001001.

1.3 Main instruments

Mouse tail suspension (homemade), electronic stopwatch, etc.

2. Grouping and administration

Since there is no relevant research literature on the treatment of depression with Bupleuri Radix and Radix Paeoniae Alba extract, the dosage of mice is determined to be 500mg/Kg.d (the total saponins of Radix Bupleuri and total saponins of Radix Paeoniae Alba) based on experience, and the positive group is given clomipramine hydrochloride Tablets, dosage 20mg/Kg, normal group was given 0.5% CMC-Na solution. The dose regimen of the drug administration group is shown in Table 8.

Table 8 Design of dosage regimen for extract administration

3. Experimental method

3.1 Mouse tail suspension test

The mice were administered by gavage continuously for 7 days. One hour after the last gavage, the tail end of the mouse was fixed on a horizontal bar at 2 cm, so that it was in an upside-down state. , Record the immobility time of the mice within 6 minutes.

3.2 Mice forced swimming test

Do a preliminary test first, put the mice into a beaker with a diameter of 18cm and a water depth of 10cm, keep the water temperature at 25°C, swim the mice for 2 minutes, then start to observe, the observation lasts for 4 minutes, within 4 minutes, the mice stop struggling in the water and show signs of life. Floating state, only small limb movements to keep the head floating on the water (immobility time), the mice whose immobility time exceeds 120 seconds or less than 60 seconds are removed. Qualified mice were randomly divided into groups and gavaged continuously for 7 days. One hour after the last gavage, the forced swimming test was performed according to the pre-test method, and the immobility time of the mice within 4 minutes was accumulated.

4. Experimental results

4.1 Effects of different formula ratios on the body weight of mice

After the last oral administration, the mice in each group were weighed, and the results are shown in Table 9. It was found that the body weight of the treatment group was lighter than that of the normal group and the positive group, except for the treatment group 1 and the treatment group 7, there were significant differences.

Table 9 The impact of different formula ratios on the body weight of mice

Note: Compared with the normal group: ^* P<0.05, ^** P<0.01

4.2 Effects of different formula ratios on the tail suspension time of mice

In the state of tail suspension, the mouse will soon appear desperate behavior, showing that it no longer struggles, and presents a continuous quiet state. It can be seen from Table 10 that, except for treatment group 9, compared with the normal group, all drug administration groups can significantly shorten the tail suspension immobility time, and treatment group 8 has the best effect.

Table 10 Effects of different formula ratios on the tail suspension time of mice

Note: Compared with the normal group: ^* P<0.05, ^** P<0.01, ^*** P<0.001

4.3 Effects of different formula ratios on the immobility time of mice forced to swim

It can be seen from Table 11 that compared with the normal group, the treatment group 3, treatment group 8 and positive group can significantly shorten the immobility time of forced swimming.

Table 11 Effects of different formula ratios on the immobility time of mice forced to swim

Note: Compared with normal group: ^* P＜0.05

5 Conclusion

Mouse tail suspension test and mouse forced swimming test are commonly used antidepressant drug screening methods, and the experimental results are relatively reliable. The experimental results showed that treatment group 8 (total saponins of Bupleuri: total saponins of paeony = 1:9) had the best antidepressant effect, and treatment group 9 (total saponins of paeony: total saponins of paeony = 0:10) had the best antidepressant effect. Difference. Therefore, although the amount of Bupleurum is small, it plays a key role. However, other formula ratios, even Bupleurum alone, have unsatisfactory antidepressant effects. It can be seen that the compatibility ratio of Bupleurum and Radix Paeoniae Alba is a key factor affecting its pharmacological effects. In the experiment, it was also found that the weight of the treatment group after administration was lighter than that of the normal group and the positive group, suggesting that it may have the function of reducing fat and losing weight.

Treat group 8 according to the best curative effect, determine that the formula ratio of Jieyu capsule is total saponins of Bupleuri: total saponins of white paeony=1:9, and the dosage of mice is 500 mg/Kg every day, i.e. total saponins of Bupleurum 50 mg/Kg, white paeony Total paeony saponins 450mg/Kg. In terms of body surface area, it is equivalent to an adult taking 2000mg of Jieyu Capsules per day, namely 200mg of total saponins of Bupleurum and 1800mg of total saponins of Paeony. The amount of crude drugs converted into crude drugs is equivalent to 4.8g of Bupleurum and 58.7g of Paeoniae Alba.

Five, the main pharmacodynamic research of composition of the present invention

1. Experimental materials

1.1 Drugs and reagents

The above-mentioned Bupleurum extract and the above-mentioned Radix Paeoniae Alba extract are suspended with 0.5% CMC-Na, homogenized by a high-speed homogenizer for 10 minutes, made into a suspension, and shaken before use;

Clomipramine hydrochloride tablets (Novartis Pharmaceuticals, batch number X0026, 25mg/tablet);

Reserpine injection (Guangdong Bangmin Pharmaceutical Factory Co., Ltd., batch number 090101, 1mg/1ml);

Norepinephrine (L-NE, SIGMA company, lot number A-9512);

Dopamine (DA, SIGMA company, lot number H8502)

5-Hydroxyindoleacetic acid (5-HIAA, SIGMA company, lot number H8876)

5-hydroxytryptamine (5-HT, SIGMA company, lot number H7752)

Acetic acid, perchloric acid, and sodium acetate are analytically pure reagents, and methanol is chromatographically pure reagents.

1.2 Experimental animals

ICR mice and SD rats are clean grade. Half male and half female, provided by Zhejiang Experimental Animal Center, experimental animal qualification certificate number: Zheyi Dongzi No. 2001001.

1.3 Main instruments

Mouse tail suspension, homemade, electric shock cage, homemade.

Animal mental behavior analyzer, NOLDUS company in the Netherlands, Ethovision3.0 analysis software.

High-performance liquid chromatography analysis system, fluorescence detector, American VARIAN company.

Automatic homogenizer, centrifuge, electronic stopwatch, electronic thermometer, etc.

2. Grouping and administration

2.1 Mouse experiment

Normal group: give 0.5% CMC-Na solution;

Composition small dose group: 250mg/kg, homogeneously suspended with 0.5% CMC-Na;

Dosage group in the composition: 500mg/kg, homogeneously suspended with 0.5% CMC-Na;

Composition high-dose group: 750mg/kg, homogeneously suspended with 0.5% CMC-Na;

Positive group: clomipramine hydrochloride tablets: 20 mg/kg, homogeneously suspended in 0.5% CMC-Na.

According to the volume of 0.1ml/10g gavage.

2.2 Rat experiment

Normal group: give 0.5% CMC-Na solution;

Model group: give 0.5% CMC-Na solution;

Composition small dose group: 125mg/kg, homogeneously suspended with 0.5% CMC-Na;

Dosage group in the composition: 250mg/kg, homogeneously suspended with 0.5% CMC-Na;

Composition high-dose group: 375 mg/kg, homogeneously suspended with 0.5% CMC-Na;

Positive group: clomipramine hydrochloride tablets: 10 mg/kg, homogeneously suspended in 0.5% CMC-Na.

According to the volume of 1ml/100g gavage.

3. Experimental method

3.1 Autonomous activity test of mice

Mice were fed Jieyu Capsules continuously for 7 days. One hour after the last gavage, they were placed in a 75×75cm special activity field, and the field was divided into 25 grids on average. After 3 minutes of adaptation, continue to observe the mice within 5 minutes For the activity situation, the animal's movement distance, the activity rate of the edge grid (that is, the percentage of the movement distance in the edge grid to the total activity distance), the number of times the two forelimbs are off the ground, the immobility time and the dwell time of the central grid are calculated with the animal mental behavior analysis software.

3.2 Mouse tail suspension test

Mice were given Jieyu Capsules for 7 days continuously. One hour after the last gavage, the mouse tail was fixed on a horizontal bar at 2 cm, so that it was in an upside-down state. The sight of the animal was recorded, and the immobility time of the mouse within 6 minutes was recorded.

3.3 Mice forced swimming test

Do a preliminary test first, put the mice into a beaker with a diameter of 18cm and a water depth of 10cm, keep the water temperature at 25°C, swim the mice for 2 minutes, and then start to observe. The duration of small limb movements to keep the head floating on the water surface (immobility time), and the mice whose immobility time exceeds 120 seconds or is less than 60 seconds are removed. Qualified mice were randomly divided into groups and gavaged continuously for 7 days. One hour after the last gavage, the forced swimming test was performed according to the pre-test method, and the immobility time of the mice within 4 minutes was accumulated.

3.4 Antireserpine experiment in rats

SD rats were given Jieyu Capsules continuously for 7 days, and 1 hour after the last gavage, subcutaneously injected reserpine 3.5mg/kg, observed the time of eyelid ptosis in rats, and measured it 2h and 18h after injection of reserpine Anal temperature.

3.5 Chronic unpredictable stress model test

水温20℃)，游泳15min训练1次并熟悉环境。然后每天灌胃给药1次，参照文献方法，按下列顺序给予不良刺激：禁食不禁水48h(2d)-电击5min(36V，10mA，上下午各1次)(1d)-禁水不禁食24h(1d)-冰水游泳5min(1d)-夹尾悬吊5min(1d)，完成1轮刺激共6d。恢复1d，第7d称体重后，每笼2只(个别3只)饲养，禁食，并喂以1％蔗糖水溶液，测定24h蔗糖水饮用量(奖赏反应)。第8d恢复正常供食和供水，第9d开始同样方法第二轮刺激。完成第2轮刺激后同法测定蔗糖水饮用量。次日，灌胃给药后30min，将大鼠置于水桶内，1min后开始记时，并用秒表记录5min内大鼠累计漂浮在水面上不动的时间，恢复一天后，将动物处死，取大脑海马组织测定单胺类神经递质(L-NE、DA、5-HIAA、5-HT)的含量。For SD rats, before the experiment, place the rats individually in a plastic bucket filled with 2/3 tap water (the height of the bucket is 55cm,

Water temperature 20°C), swimming for 15 minutes for training once and getting familiar with the environment. Then intragastric administration once a day, according to the method in the literature, give adverse stimuli in the following order: fasting without water for 48 hours (2d)-electric shock for 5min (36V, 10mA, once in the morning and afternoon) (1d)-without water Eat for 24h (1d)-swim in ice water for 5min(1d)-tail suspension for 5min(1d), complete one round of stimulation for a total of 6d. After recovering for 1 day and weighing on the 7th day, 2 animals (3 individuals) were fed in each cage, fasted, and fed with 1% sucrose aqueous solution, and the drinking amount of sucrose water was measured for 24 hours (reward response). On the 8th day, the normal food and water supply was resumed, and on the 9th day, the second round of stimulation was started in the same way. After completing the second round of stimulation, the consumption of sucrose water was determined in the same way. On the next day, 30 minutes after intragastric administration, the rats were placed in a water bucket, and the time was started after 1 minute, and a stopwatch was used to record the accumulated floating time of the rats on the water surface within 5 minutes. After one day of recovery, the animals were put to death and taken. The contents of monoamine neurotransmitters (L-NE, DA, 5-HIAA, 5-HT) were determined in the hippocampal tissue of the brain.

Since monoamine neurotransmitters are easily oxidized, sample processing must be carried out under low temperature and dark conditions. Take out the hippocampus as soon as possible on the ice platform, rinse with normal saline, remove the blood, dry it with filter paper, and weigh it. Add about 1.0ml of ice-cold 0.4mol/L perchloric acid solution to the hippocampus of each rat, homogenize with a high-speed homogenizer in an ice bath, transfer the hippocampal tissue homogenate into a 2.0ml Ependet centrifuge tube, and place in a refrigerated centrifuge Centrifuge at 12000r/min for 15 minutes, transfer the supernatant to a 1.0ml volumetric flask, and dilute to the mark with 0.4mol/L perchloric acid solution to obtain a sample solution for later use.

Chromatographic conditions: Chromatographic column: YMC-C18 column (250×4.6mm 5μm). The mobile phase is 0.1mol L-1 sodium acetate (containing 0.1mmol L-1 EDTA-2Na), adjusted to pH 5.1 with Hac, filtered through a 0.45μm microporous membrane, and degassed by ultrasonic. Mobile phase B, methanol as mobile phase A, isocratic elution at a ratio of 9:1, flow rate 1.0ml/min, injection 10 μL, emission wavelength 330nm, excitation wavelength 290nm. Column temperature: 30°C.

4 Experimental results

4.1 The influence of composition on normal mouse autonomic activity

It can be seen from Table 12 that, except that the number of forelimbs in the low-dose group is lower than that of the normal group, the composition has no significant impact on the indicators of normal mouse autonomic activity, suggesting that the composition is not a central nervous system stimulant.

The influence of table 12 compositions on normal mouse autonomic activity ( n=10)

Note: Compared with normal group: ^* P＜0.05

4.2 Effect of the composition on the tail suspension time of normal mice

It can be seen from Table 13 that the positive group, the low-dose group and the middle-dose group of the composition can significantly reduce the tail suspension time compared with the normal group.

The influence of table 13 compositions on mouse tail suspension time

Note: Compared with normal group: ^* P＜0.05

4.3 Effect of the composition on the immobility time of mice forced to swim

It can be seen from Table 14 that the positive group, the low-dose group and the middle-dose group of the composition, compared with the normal group, can significantly reduce the immobility time of forced swimming.

The impact of table 14 composition on the immobility time of mice's forced swimming

Note: Compared with normal group: ^* P＜0.05

4.4 Effects of Compositions on Reserpine Antagonism in Rats

Reserpine has the effect of emptying the monoamine neurotransmitter in the brain, can cause ptosis of the eyelids in rats, and cause a significant drop in body temperature. It can be seen from Table 15 that, compared with normal rats, the composition can significantly prolong the occurrence time of eyelid ptosis after the rats are injected with reserpine, and presents a certain dose-effect relationship. At the same time, compared with the model group, the composition can significantly restore the body temperature of rats.

The influence of table 15 composition on rat antagonism reserpine

Note: Compared with the model group: ^* P<0.05, ^** P<0.01, ^*** P<0.001

Compared with normal group: #P＜0.05

4.5 Effects of Composition on Chronic Depression Model Rats

After the combination of various adverse stimuli, the rats' body weight growth rate decreased significantly, their general condition was slightly worse, their fur was fluffy, and their activities decreased. It can be seen from Table 16 that in the 7 days of the first round of stimulation, the weight gain was significantly slowed down compared with the normal group, and the high-dose group even lost weight. During the 7 days of the second round of stimulation, the weight gain of each modeling group was improved compared with the first round, but still lower than that of the normal group. Compared with the model group, the body weight of the rats in the administration group was significantly lower than that of the model group in the first round of stimulation, and there was no significant difference between the other groups and the model group. After the completion of the second round of stimulation, there was no significant difference in the body weight of the rats in the administration group compared with the model group.

The amount of sugar water consumed reflects the degree of response of animals to rewards, and the response of depression model animals to rewards decreased, and the amount of sugar water consumed was significantly reduced. It can be seen from Table 17 that the water consumption of the rats in the model group in this experiment was only 75% and 66% of the normal group after the first and second rounds of stimulation. The composition can obviously increase the drinking amount of sugar water of model animals and improve the response to reward, and there is a certain dose relationship.

When a rat enters a limited space to make it swim, it swims desperately at the beginning and tries to escape. After repeated efforts and failures, it appears a desperate behavior, which is manifested as a floating state. In fact, the animal gives up the hope of escape, which belongs to the behavior of despair and immobility. The length of the movement time can represent the degree of despair (depression) of the rat. It can be seen from Table 18 that the swimming immobility time of the model group rats is significantly increased compared with the normal group rats, and the composition has obvious antagonism to the swimming desperate behavior of the chronic depression model rats, and can significantly shorten the swimming immobility time, the dose-effect relationship clear. The immobility time of swimming in the high-dose group was reduced by 58% compared with the model group, and the immobility time of swimming in the small and medium dose group was also significantly shortened. It can be seen from Table 19 that Jieyu Capsules can significantly improve the levels of monoamine neurotransmitters (L-NE, DA, 5-HIAA, 5-HT) in the hippocampus of chronic depression model animals, and preliminarily clarified the improvement of monoamine neurotransmitters in the brain. Transmitter level is one of the antidepressant mechanism of Jieyu Capsules.

Table 16 composition influences the body weight of chronic depression model rats

Note: Compared with normal group: ##P＜0.01, ###P＜0.001

Compared with the model group: ^** P＜0.01

Table 17 Effect of composition on reward response in chronic depression model rats

Note: Compared with normal group: #P＜0.05, ##P＜0.01

Compared with the model group: ^* P<0.05, ^** P<0.01

Table 18 Effects of Compositions on Forced Swimming Immobility Time in Chronic Depression Model Rats

Note: Compared with normal group: #P＜0.05

Compared with the model group: ^* P<0.05, ^*** P<0.001

Table 19 Effects of compositions on monoamine neurotransmitters in the hippocampus of chronic depression model rats

Note: Compared with normal group: #P<0.05, ##P<0.01, ###P<0.001

Compared with the model group: ^* P<0.05, ^** P<0.01, ^*** P<0.001

5 Summary of Pharmacodynamic Tests

In the experiment, recognized antidepressant animal test methods such as mouse tail suspension test, mouse forced swimming test, rat antagonism reserpine test, and rat chronic unpredictable stress model test were used to study the pharmacodynamics of the composition. The Animal Psychobehavioral Analysis System observes the effect of compositions on the voluntary activities of animals. The results confirm that the composition can obviously shorten the immobility time of the tail suspension of mice and the immobility time of mice forced to swim. For rats modeled with high-dose reserpine, it can significantly prolong the appearance time of eyelid ptosis, and obviously antagonize the drop in body temperature caused by reserpine. For chronic depression model rats, the composition can obviously improve the reward response of the model rats to sucrose water, and can obviously shorten the immobility time of the model rats in forced swimming, and its effect is close to that of clomipramine hydrochloride. Through the open field test, it is confirmed that the composition has no obvious influence on normal animal autonomic activity, indicating that the composition is not a central nervous system excitatory drug. By measuring the content of monoamine neurotransmitters in the hippocampal tissue of the chronic depression model rats, it was confirmed that the composition can significantly increase the levels of L-NE, DA, 5-HIAA, and 5-HT in the hippocampus of the model animals, and preliminarily clarified the Improving the level of monoamine neurotransmitters in the brain is one of the antidepressant action mechanisms of the composition.

In the pharmacodynamics test, no correlation between dosage and curative effect was found in the tail suspension test and forced swimming test of normal mice, and the middle dose group had the best effect. However, in the rat antagonism reserpine experiment that needs to be modeled and the rat chronic unpredictable stress model experiment, the composition shows an obvious dose-effect relationship. It may be related to the fact that the composition is more likely to exert its medicinal effect in a pathological state. At the same time, it is also found that the composition also has a certain inhibitory effect on the weight gain of animals, and whether it will affect the lipid metabolism of animals remains to be further studied.

Pharmacodynamic experiments show that the small dose group of the composition has obvious antidepressant pharmacological effects, which is equivalent to an adult taking 1000 mg of the composition per day, that is, 100 mg of total saponins of Bupleuri and 900 mg of total saponins of paeony, converted into crude drugs The amount is equivalent to 2.4g of Bupleurum and 29.4g of Radix Paeoniae Alba. Mechanism studies have shown that the low-dose group has no significant effect on increasing the neurotransmitters in the hippocampus of model animals, suggesting that the antidepressant composition of the composition may have other mechanisms of action, and multiple targets work together.

Six, the toxicology research of composition of the present invention:

1. Acute toxicity test: because the toxicity of the composition is very low, the LD50 cannot be measured, and the maximum administration dose of the mouse is 7.5g/kg (composition/body weight), which is equivalent to the dosage for clinical adults (according to the body surface area) Conversion) 1000 times, continuous observation for 10 days, no obvious toxic reaction.

2. Long-term toxicity test: the composition was administered to rats, the large dose group 7.5g/kg is 1000 times the clinical adult dose, the medium dose 2.5g/kg is 333 times the clinical adult dose, and the small dose 0.75g/kg It is 100 times of the clinical dosage for adults. After continuous intragastric administration for 6 months, no rats were found to die under high-dose conditions, and no obvious toxic reactions were seen, indicating that the composition is relatively safe.

The composition of the present invention is composed of Radix Bupleurum and Radix Paeoniae Alba. In the formula, Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Bupleuri to Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Bupleuri and Radix Radix Radix Radix Radix Radix Radix Paeoniae. Yin blood is good at growing, and it can soften the liver and calm the liver, relieve spasm and relieve pain, and clear away asthenic heat. The combination of the two medicines, Bupleurum to obtain the softness of Radix Paeoniae Alba, promotes liver qi without excessive dredging and exhausting the liver body; Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Radix Paeoniae Alba nourishes liver blood without causing stagnation of qi and yin, hindering the function of the liver. It plays the functions of soothing the liver, relieving depression and relieving pain from the outside and inside. It can not only soothe the liver and relieve stagnation, so as to treat the insufficiency of the liver, but also soften the liver and replenish yin to nourish the liver and body. It is the best combination for both body and function.

The invention extracts total saponins of Bupleuri and total saponins of paeony through modern pharmaceutical technology, and prepares Jieyu capsules according to a certain compatibility ratio, has clear medicinal effective parts and clear curative effects, and has no obvious toxic and side effects. The preparation method of the composition of the invention is stable in process, feasible in operation, high in extraction rate, high in purity, less in environmental pollution, suitable for industrial production, and capable of achieving better social and economic benefits.

Claims (5)

1. the Chinese medicine composition of an antidepressant effect is made by the extract of following weight portion: Radix Paeoniae Alba extract 1-10 part, Radix Bupleuri extract 1-10 part.

2. Chinese medicine composition according to claim 1 is characterized in that, described Radix Paeoniae Alba extract is 9 parts, and Radix Bupleuri extract is 1 part.

3. a Chinese medicine preparation is made by Chinese medicine composition as claimed in claim 1 or 2, and its dosage form is any one in oral liquid, tablet, capsule, drop pill and the granule.

4. Chinese medicine preparation, starch by weight such as Chinese medicine composition addings as claimed in claim 2, mix homogeneously, fill makes in capsule, content of paeoniflorin is no less than 7.50% in the capsule, the content of Radix Paeoniae Alba total saponins is no less than 25.0%, and the content of saikoside d is no less than 0.50%, and the content of Radix Bupleuri total saponin is no less than 2.50%.

5. the preparation method of a Chinese medicine composition is characterized in that, comprises the steps:

1) preparation Radix Paeoniae Alba extract: get Radix Paeoniae Alba decoction pieces, add 8 times of amount 70% ethanol respectively, extract 3 times, each 2 hours, gained extracting solution decompression recycling ethanol was concentrated into 2.0g crude drug/ml, get the concentrated solution of 1BV volume, cross the AB-8 macroporous adsorbent resin with 1BV/ hour flow velocity, leave standstill 2 hours after, clean to effluent colourless with deionized water with 1BV/ hour flow velocity, carry out eluting with 10BV 70% ethanol with 2BV/ hour speed, collect eluent, decompression recycling ethanol is concentrated into extractum, 60 ℃ of vacuum dryings, take out, pulverize, obtain Radix Paeoniae Alba extract;

2) preparation Radix Bupleuri extract: get the Radix Bupleuri decoction pieces, add 80% ethanol that 10 times of amounts contain 1.0% sodium carbonate respectively, heating and refluxing extraction 3 times, each 1.5 hours, merge extractive liquid; The extracting solution decompression recycling ethanol of gained, be concentrated into 0.2g crude drug/ml, measure the 1BV extracting solution, cross the AB-8 macroporous adsorbent resin with 1BV/ hour flow velocity, after leaving standstill 2 hours,, discard eluent with the flow velocity eluting of deionized water 10BV with 1BV/ hour, reuse 20% ethanol 10BV cleans with 1BV/ hour flow velocity, discard cleanout fluid,, collect eluent with the speed eluting of 70% ethanol 10BV with 2BV/ hour, decompression recycling ethanol, be concentrated into extractum, 60 ℃ of vacuum dryings take out, pulverize, obtain Radix Bupleuri extract;

3) preparation compositions: by Radix Paeoniae Alba extract: Radix Bupleuri extract is 9: 1 a weight proportion, takes by weighing above-mentioned two kinds of extracts, and mix homogeneously is made the described Chinese medicine composition of claim 2.